Hemoglobin (Hb) is an oxygen-carrying protein enriched in red blood cells (erythrocytes) that delivers oxygen to body tissues via blood flow through the circulatory system. The oxygen-carrying protein comprises four associated polypeptide chains (two alpha chains and two beta chains), and bears a group known as heme whose iron atom temporarily binds to oxygen in the lungs and releases it throughout the body. Hemoglobin can also bind carbon monoxide (CO) to form (carbonmonoxy)hemoglobin (HbCO), which can reduce the total amount of Hb that is available to deliver oxygen to the body.
Hypoxia is common in cancers and can lead to ionizing radiation and chemotherapy resistance by depriving tumor cells of the oxygen essential for the cytotoxic activities of these agents. Hypoxia may also reduce tumor sensitivity to radiation therapy and chemotherapy through one or more indirect mechanisms that include proteomic and genomic changes.
Hemoglobin extracted from red blood cells has been used as a blood substitute for oxygen delivery to hypoxic tissues. However, the use of unmodified cell-free Hb purified from red blood cells can suffer from several limitations, such as contamination, supply limitations, an increase in oxygen affinity due to loss of the cofactor 2,3-disphosphoglycerate (2,3-DPG), and dissociation of Hb tetramers into aP dimers, which are cleared by renal filtration and can cause long-term kidney damage.
The development of improved recombinant hemoglobins can solve some or all of the above-mentioned problems. However, existing recombinant hemoglobins can suffer from low oxygen carrying capacity, and the existing methods for preparing recombinant hemoglobins are laborious and can suffer from poor soluble yields of hemoglobin with high percentages of impurities. For example, protoporphyrin-IX (PPIX), a common byproduct in conventional recombinant hemoglobin production, can impair the overall oxygen carrier function of the recombinant hemoglobins.
Existing methods for preparing recombinant hemoglobins generally include a heat treatment step to remove impurities, such as PPIX. However, the incorporation of a heat treatment step into the preparation method can increase cost and reduce yield of the protein product. In addition, in order to maintain the stability of the recombinant hemoglobins during the heat treatment and downstream processes thereof, CO is required for the recombinant hemoglobins. However, since CO is undesired in the final product, a CO removal step is usually required for existing preparation methods, which also makes the existing methods costly and time-consuming.
Therefore, there is a need for improved recombinant hemoglobins with high oxygen carrying capacity, and there is also a need for simplified methods for preparing recombinant hemoglobins with high yield and high purity.